We now have the capability to reprogram a somatic cell to a pluripotent state, a so-called induced pluripotent stem cells (iPSCs), which have many of the attributes of embryonic stem cells (ESCs). These include self- renewal and the ability to be directed to the three germ layers. Importantly, iPSCs retain the genetic composition of parental cells, and as a consequence their potential utility as autologous donors for cell therapy and in vitro disease modeling has been recognized. The epigenetic states in iPSCs are similar to ESCs but they differ, especially with respect to methylation. These include differentially methylated regions and incomplete erasure of parental DNA methylation (epigenetic memory), these in turn have repercussions in differentiation potential resulting in unpredictable behavior o iPSC-derivatives. To safeguard against these undesirable side-effects it is crucial to investigate the reprogramming process at the epigenetic level, with the ultimate goal of generating desirable iPSCs. Among the proteins involved in DNA methylation and demethylation, is the hemi-methylated DNA binding protein NP95, which increases reprogramming efficiency and can substitute for c-Myc. Accompanied with these observations, NP95 increases H3K4me3 as well as hydroxymethylcytosine (hmC). These implicate a novel function for NP95 in transcriptional activation during reprogramming, contrary to reported involvement in maintenance of methylated DNA and heterochromatic regions. This proposal will address a number of gaps in our knowledge of reprogramming, by providing crucial insight into the function of NP95. This will be achieved by implementing these specific aims: (1) Determine whether NP95 increases SET1 activity. We will establish whether NP95 stabilizes the SET1 complex or directly activates the catalytic activity of Set1/COMPASS complex and identify the domains critical for SET1 activity. (2) Determine whether NP95 recruits Set1a for H3K4me3 marks. We will assess the ability of NP95 to recruit Set1 complex to targets and mediates euchromatin gene activation. This will be achieved with ChIP-seq against NP95, Set1 and H3K4me3. The TTD domain of NP95 is known to interact with modified histone H3. It will be interrogated to assess its role in H3K4me3 formation during reprogramming. (3) Determine whether NP95 reads hmC for H3K4me3 formation. In ESCs, hmC marks the loci of active genes. The hmC marks are produced by TET proteins induced during the reprogramming process. Recent studies have shown that NP95 binds to hmC as well as mC. We will confirm these observations and further develop this by examining the formation of mC and hmC by NP95 during reprogramming. This proposal will significantly impact on the reprogramming field by providing for the first time a detailed study of molecular events during reprogramming. Additionally we will dissect the novel function of NP95 in transcriptional activation during reprogramming and pluripotent stem cells. Ultimately our data will be critical in generating clinically safe, appropriately reprogrammed iPSCs for cell therapy and disease modeling.